There are several strategies that can be deployed to increase target protein identifications: utilize post-translational modification (PTM) specific affinity binding, such as phosphopeptide enrichment, or through fractionation, such as high pH reversed-phase chromatography which separates peptides by hydrophobicity.

The novel Thermo Scientific™ ActivX™ probes and enrichment kits enable the specific targeting of several enzyme classes, including kinases, GTPases, and serine hydrolases, which are covalently labeled in the active site at the protein level and enriched after digestion.

Number of Phosphates per Peptide

1

2

3

4

5

6

Total

TiO2

492

103

8

4

0

1

608

Fe-NTA IMAC

234

34

216

3

1

0

488

Overlap

155

0

1

0

0

0

156

Selective enrichment of singly and multiply phosphorylated phosphopeptides with TiO2 and Fe-NTA IMAC. Average phosphopeptide enrichment results from duplicate experiments showing the number ofphosphopeptides containing one or more phosphate per peptide enriched using either method. Peptide spectrum summary results were exported from Proteome Software Scaffold 3.0 and analyzed with Microsoft™ Excel™ Software.